【摘要】：背景和目的:齲病和牙周病是臨床常見疾病、多發(fā)病,往往造成牙髓壞死和牙槽骨吸收以致牙齒缺失�，F(xiàn)有的診療技術(shù)往往無法真正意義上實(shí)現(xiàn)牙髓、牙周組織的生理性和功能性再生。因此,在當(dāng)代口腔醫(yī)學(xué)領(lǐng)域,牙髓和牙周組織再生治療仍然是學(xué)者們研究的熱點(diǎn)。隨著組織工程技術(shù)的不斷發(fā)展,外源性干細(xì)胞植入和自體干細(xì)胞歸巢療法在許多組織器官再生的實(shí)驗(yàn)研究中已經(jīng)被廣泛開展。然而,由于牙齒解剖結(jié)構(gòu)的特殊性,學(xué)者們往往難以將干細(xì)胞有效的遷移并富集在牙髓或者牙周組織,同時(shí)在進(jìn)行干細(xì)胞治療過程中也無法對(duì)植入的干細(xì)胞進(jìn)行示蹤觀察,這些都對(duì)牙髓和牙周組織原位再生帶來了極大的挑戰(zhàn)。近年來出現(xiàn)的磁靶向技術(shù)可以極大的促進(jìn)植入的干細(xì)胞在靶器官的遷移和定植,同時(shí)與之相輔相成的臨床MRI檢測(cè)手段還可以對(duì)植入細(xì)胞進(jìn)行無創(chuàng)的示蹤觀察,這些都為學(xué)者進(jìn)行牙髓和牙周組織再生指明了新的研究方向。在本試驗(yàn)中,我們首先選擇了一種新的自帶熒光且無需轉(zhuǎn)染劑的SPIO(Molday ION Rhodamine-B,MIRB?)對(duì)人牙髓干細(xì)胞進(jìn)行了標(biāo)記,探討了對(duì)其活力、增殖、分化等一系列生物學(xué)影響,進(jìn)一步通過MRI技術(shù)對(duì)裸鼠皮下植入的細(xì)胞進(jìn)行無創(chuàng)示蹤觀察。接下來我們用SPIO標(biāo)記了大鼠骨髓間充質(zhì)干細(xì)胞,在體外磁場(chǎng)環(huán)境下誘導(dǎo)細(xì)胞遷移,同時(shí)構(gòu)建大鼠牙槽骨缺損模型,從靜脈注射以及局部注射兩種細(xì)胞輸入途徑進(jìn)行研究,觀察體內(nèi)環(huán)境下磁靶向技術(shù)在促進(jìn)干細(xì)胞在牙槽骨缺損處的遷移和定植。本實(shí)驗(yàn)實(shí)現(xiàn)了MRI活體示蹤觀察植入的干細(xì)胞,還豐富了磁靶向技術(shù)在牙周組織再生中的運(yùn)用,為未來牙周再生干細(xì)胞療法提供了新思路。結(jié)果如下:第一部分MIRB標(biāo)記對(duì)h DPSCs的生物學(xué)影響以及體內(nèi)體外MRI成像1.人牙髓干細(xì)胞(h DPSCs)培養(yǎng)和鑒定本研究采用酶消化法/組織塊法從成人第三磨牙牙髓組織中分離培養(yǎng)人DPSCs,利用單細(xì)胞克隆法純化細(xì)胞,通過體外成骨、成脂誘導(dǎo)分化以及流式細(xì)胞儀鑒定確定了其干細(xì)胞屬性。2.MIRB標(biāo)記h DPSCs分別用終濃度為12.5,25,50,100μg Fe/m L的SPIO(MIRB?)標(biāo)記h DPSCs,利用激光共聚焦、普魯士藍(lán)染色光鏡觀察以及透射電鏡等手段觀察了SPIO納米顆粒在h DPSCs細(xì)胞內(nèi)的分布情況,分光光度計(jì)測(cè)量了各組細(xì)胞內(nèi)鐵含量。結(jié)果發(fā)現(xiàn),MIRB可以不需要轉(zhuǎn)染劑即可高效的標(biāo)記h DPSCs,各種濃度標(biāo)記后對(duì)細(xì)胞的形態(tài)沒有影響,且隨著標(biāo)記濃度的升高細(xì)胞內(nèi)鐵含量也逐漸上升。3.MIRB對(duì)h DPSCs體外生物學(xué)特性的影響采用臺(tái)盼藍(lán)排斥實(shí)驗(yàn)、CCK8實(shí)驗(yàn)、流式細(xì)胞儀、體內(nèi)外成骨誘導(dǎo)等手段分別檢測(cè)了MIRB標(biāo)記對(duì)h DPSCs的活力、增殖、干細(xì)胞表型、細(xì)胞周期、凋亡以及成骨分化的影響。結(jié)果發(fā)現(xiàn),12.5-50μg/m L的MIRB標(biāo)記不影響h DPSCs的活力且可以促進(jìn)細(xì)胞增殖,而100μg/m L則對(duì)細(xì)胞有毒性,最佳標(biāo)記濃度為12.5μg/m L;12.5μg/m L的MIRB標(biāo)記不影響h DPSCs的干細(xì)胞表型;12.5μg/m L-50μg/m L的MIRB標(biāo)記可以加快細(xì)胞周期而對(duì)細(xì)胞凋亡無影響;12.5μg/m L-50μg/m L的MIRB對(duì)h DPSCs成骨分化無影響。4.MIRB標(biāo)記牙髓干細(xì)胞的體外、體內(nèi)MRI成像觀察通過MRI檢測(cè)手段,體外觀察了MIRB標(biāo)記的h DPSCs細(xì)胞團(tuán)塊,體內(nèi)觀察了MIRB標(biāo)記的細(xì)胞膜片在裸鼠皮下的生長情況。體外結(jié)果發(fā)現(xiàn),12.5g/m L的MIRB標(biāo)記1×10~5個(gè)細(xì)胞無法在MRI清晰顯影,而用12.5g/m L-100g/m L的MIRB標(biāo)記1×106個(gè)細(xì)胞均可清晰顯影。體內(nèi)結(jié)果發(fā)現(xiàn),MIRB標(biāo)記的h DPSCs細(xì)胞膜片在MRI下可以清晰呈負(fù)性對(duì)比影像,且隨著植入時(shí)間延長,無信號(hào)影像所占比率也逐漸下降。組織學(xué)檢查發(fā)現(xiàn),膜片內(nèi)普魯士藍(lán)陽性染色細(xì)胞數(shù)量也隨著時(shí)間的延長而逐漸下降。第二部分SPIO對(duì)大鼠骨髓間充質(zhì)干細(xì)胞的生物學(xué)影響1.大鼠骨髓間充質(zhì)干細(xì)胞(r BMMSCs)培養(yǎng)和鑒定成功采用全骨髓貼壁法培養(yǎng)了大鼠骨髓間充質(zhì)干細(xì)胞(r BMMSCs)。通過克隆形成實(shí)驗(yàn)、干細(xì)胞表型測(cè)定以及多向分化能力檢測(cè)確定其干細(xì)胞屬性。2.Resovist標(biāo)記r BMMSCs分別用終濃度為25、50、100g/m L的SPIO(Resovist?)標(biāo)記r BMMSCs,利用普魯士藍(lán)染色光鏡觀察以及透射電鏡等手段觀察了Resovist納米顆粒在r BMMSCs細(xì)胞內(nèi)的分布情況。結(jié)果發(fā)現(xiàn),Resovist同樣可以不需要轉(zhuǎn)染劑即可高效的標(biāo)記r BMMSCs。3.Resovist對(duì)r BMMSCs生物學(xué)特性的影響采用臺(tái)盼藍(lán)排斥實(shí)驗(yàn)、CCK8實(shí)驗(yàn)、流式細(xì)胞儀、體外成骨誘導(dǎo)等手段分別檢測(cè)了Resovist標(biāo)記對(duì)r BMMSCs的活力、增殖、細(xì)胞周期、凋亡以及成骨分化的影響。結(jié)果發(fā)現(xiàn),25-50g/m L的Resovist對(duì)r BMMSCs活力沒有影響,而100g/m L對(duì)細(xì)胞有毒性;25g/m L不影響細(xì)胞增殖、細(xì)胞周期以及凋亡,而50-100g/m L則抑制細(xì)胞增殖、減緩細(xì)胞周期并促進(jìn)細(xì)胞凋亡,說明25g/m L的Resovist是標(biāo)記r BMMSCs的合適濃度;25g/m L的Resovist對(duì)r BMMSCs的成骨分化有促進(jìn)作用。第三部分SPIO標(biāo)記的大鼠骨髓間充質(zhì)干細(xì)胞在體外體內(nèi)磁場(chǎng)環(huán)境下遷移和定植的實(shí)驗(yàn)研究1.體外磁場(chǎng)環(huán)境下SPIO標(biāo)記的r BMMSCs遷移實(shí)驗(yàn)利用Transwell小室,在體外磁場(chǎng)環(huán)境下觀察SPIO標(biāo)記的r BMMSCs的穿膜能力。結(jié)果發(fā)現(xiàn),磁場(chǎng)可以明顯促進(jìn)SPIO標(biāo)記的細(xì)胞穿膜。另外,我們?cè)谂囵B(yǎng)皿底部正中心粘貼磁鐵,將SPIO標(biāo)記的r BMMSCs接種后觀察細(xì)胞在培養(yǎng)皿內(nèi)的分布情況,發(fā)現(xiàn)細(xì)胞圍繞磁鐵周圍呈圓環(huán)狀分布。2.體內(nèi)磁場(chǎng)環(huán)境下SPIO標(biāo)記的r BMMSCs向牙槽骨缺損處的磁靶向遷移或定植構(gòu)建大鼠牙槽骨缺損模型,分別從靜脈途徑和局部注射途徑輸入SPIO標(biāo)記的干細(xì)胞。結(jié)果發(fā)現(xiàn),從靜脈輸入的干細(xì)胞大部分被脾臟內(nèi)巨噬細(xì)胞所吞噬,在牙槽骨缺損處未見到遠(yuǎn)距離遷移的干細(xì)胞,而局部注射標(biāo)記后的干細(xì)胞由于有磁場(chǎng)作用可以大量聚集在牙槽骨缺損處。結(jié)論:1.第一部分研究結(jié)果表明,目前市售的最新的納米鐵顆粒MIRB可以無需添加額外的轉(zhuǎn)染劑即可高效標(biāo)記人牙髓干細(xì)胞;12.5g/m L-50g/m L的MIRB標(biāo)記人牙髓干細(xì)胞是安全可靠的;MRI可以對(duì)植入體內(nèi)的h DPSCs細(xì)胞膜片進(jìn)行無創(chuàng)實(shí)時(shí)觀察,是干細(xì)胞移植治療中進(jìn)行細(xì)胞示蹤安全有效的手段,為未來利用牙髓干細(xì)胞移植進(jìn)行牙髓原位再生提供了實(shí)驗(yàn)依據(jù)。2.第二部分研究結(jié)果發(fā)現(xiàn),德國Schering的納米鐵顆粒Resovist同樣可以無需轉(zhuǎn)染劑即可高效標(biāo)記大鼠骨髓間充質(zhì)干細(xì)胞;25g/m L的Resovist不影響大鼠骨髓間充質(zhì)干細(xì)胞的增殖和活力,50-100g/m L的Resovist則抑制r BMMSCs增殖、減緩細(xì)胞周期并促進(jìn)細(xì)胞凋亡,說明25g/m L的Resovist是標(biāo)記r BMMSCs的合適濃度;25g/m L的Resovist對(duì)r BMMSCs的成骨分化有促進(jìn)作用,其具體機(jī)制還需進(jìn)一步深入探究。3.第三部分研究結(jié)果表明,體外磁場(chǎng)環(huán)境下可以完美模擬出標(biāo)記了SPIO的干細(xì)胞在磁場(chǎng)作用下發(fā)生靶向遷移;體內(nèi)環(huán)境下,構(gòu)建了牙槽骨缺損的磁靶向誘導(dǎo)細(xì)胞歸巢實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)遠(yuǎn)距離磁靶向誘導(dǎo)SPIO標(biāo)記的干細(xì)胞歸巢至牙槽骨缺損處沒有得到理想的預(yù)期效果,而磁場(chǎng)促進(jìn)了局部注射的SPIO標(biāo)記的干細(xì)胞在牙槽骨缺損處的定植。定植的干細(xì)胞是否可以在骨缺損處繼續(xù)增殖、分化為新的骨細(xì)胞并繼而參與牙槽骨的重建尚需要進(jìn)一步更深入的研究去證實(shí)。
[Abstract]:BACKGROUND AND OBJECTIVE: Dental caries and periodontal diseases are common clinical diseases, frequently occurring, often resulting in pulp necrosis and alveolar bone resorption, resulting in tooth loss. Existing diagnostic and therapeutic techniques often fail to truly achieve the physiological and functional regeneration of pulp and periodontal tissue. With the development of tissue engineering technology, exogenous stem cell transplantation and autologous stem cell homing therapy have been widely used in many experimental studies of tissue and organ regeneration. In-situ regeneration of dental pulp and periodontal tissues is challenged by the fact that implanted stem cells can not be traced during stem cell therapy, and magnetic targeting technology can greatly promote the migration and colonization of implanted stem cells in target organs. In this study, we first selected a new self-fluorescent SPIO (Molday ION Rhodamine-B, MIRB?) for human dental pulp stem. Then we used SPIO to label rat bone marrow mesenchymal stem cells to induce cell migration in vitro and construct rat alveolar bone defect. In this study, we observed the effect of magnetic targeting technique on the migration and colonization of stem cells in the alveolar bone defect in vivo. MRI in vivo was used to observe the implanted stem cells and enrich the application of magnetic targeting technique in periodontal tissue regeneration. The results are as follows: Part 1: Biological effects of MIRB labeling on H DPSCs and MRI imaging in vivo and in vitro 1. Human dental pulp stem cells (h DPSCs) culture and identification This study used enzymatic digestion/tissue block method to isolate and culture human DPSCs from adult third molar pulp tissue, using single cell gram. MiRB labeled h DPSCs were labeled with SPIO (MIRB?) of 12.5,25,50,100 UG Fe/ml at the final concentration, respectively. Confocal laser, Prussian blue staining light microscopy and transmission electron microscopy were used to observe SPIONA. Distribution of rice granules in H DPSCs cells was measured by spectrophotometer. The results showed that MIRB could efficiently label h DPSCs without transfection. The morphology of cells was not affected by different concentrations of MIRB, and the content of iron in cells increased gradually with the increase of the concentration of MIRB. The effects of MIRB on the viability, proliferation, stem cell phenotype, cell cycle, apoptosis and osteogenic differentiation of H DPSCs were investigated by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenic induction in vitro and in vivo. The optimal labeling concentration was 12.5 ug/ml; the 12.5 ug/ml MIRB labeling did not affect the stem cell phenotype of H DPSCs; the 12.5 ug/ml-50 ug/ml MIRB labeling could accelerate the cell cycle but had no effect on the cell apoptosis; the 12.5 ug/ml-50 ug/ml MIRB had no effect on the osteogenic differentiation of H DPSCs. RB labeled dental pulp stem cells in vitro, in vivo MRI imaging observation through MRI detection, in vitro observation of MIRB labeled h DPSCs cell mass, in vivo observation of MIRB labeled cell membrane in nude mice subcutaneous growth. In vitro, 12.5g/ml of MIRB labeled 1 *105 cells can not be clearly developed in MRI, but with 12.5g/ml-100g/m. In vivo, MIRB-labeled h DPSCs showed a clear negative contrast image, and the proportion of non-signal images decreased with the implantation time. Histological examination showed that the number of Prussian blue positive cells in the membrane also decreased with the implantation time. The second part is the biological effects of SPIO on rat bone marrow mesenchymal stem cells (BMMSCs). The culture and identification of rat bone marrow mesenchymal stem cells (r BMMSCs) were successfully carried out by whole bone marrow adherence method. The phenotype of stem cells and the ability of multi-directional differentiation were determined by clone formation test. Resovist-labeled R BMMSCs were labeled with SPIO (Resovist?) of 25,50,100 g/ml, respectively. The distribution of Resovist nanoparticles in R BMMSCs was observed by Prussian blue staining and transmission electron microscopy. The effects of Resovist on the biological characteristics of R BMMSCs were studied by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenesis induction in vitro. The results showed that the effect of Resovist on the activity, proliferation, cell cycle, apoptosis and osteogenic differentiation of R BMMSCs was 25-50g/ml. BMMSCs activity was not affected, but 100g/ml was toxic to cells; 25g/ml did not affect cell proliferation, cell cycle and apoptosis, while 50-100g/ml inhibited cell proliferation, slowed cell cycle and promoted cell apoptosis, suggesting that 25g/ml of Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/ml of Roesovist could promote osteogenic differentiation of R BMMSCs. Part 3. Experimental study on the migration and colonization of SPIO-labeled rat bone marrow mesenchymal stem cells in vitro and in vivo magnetic field 1. The migration of SPIO-labeled R BMMSCs in vitro magnetic field was studied by using Transwell chamber. The permeability of SPIO-labeled R BMMSCs in vitro magnetic field was observed. In addition, the distribution of SPIO-labeled R BMMSCs in the culture dish was observed after inoculation. The cells were circularly distributed around the magnets. 2. The magnetic targeted migration or colonization of SPIO-labeled R BMMSCs to the alveolar bone defect was observed in vivo. SPIO-labeled stem cells were injected into alveolar bone defect model of rats through intravenous route and local injection route respectively. Conclusion: 1. The results of the first part of the study show that the latest nano-iron particles MIRB on the market can effectively label human dental pulp stem cells without additional transfectants; 12.5g/ml-50g/ml MIRB labeling human dental pulp stem cells is safe and reliable; MRI can be used to label human dental pulp stem cells in vivo; 2. Non-invasive real-time observation of PSCs cell membrane is a safe and effective method for cell tracing in stem cell transplantation. It provides an experimental basis for in-situ regeneration of dental pulp using dental pulp stem cell transplantation in the future. 2. The results of the second part show that the Resovist of Schering nanoparticles can also be high without transfection agent. Rat bone marrow mesenchymal stem cells were effectively labeled; 25g/m L Roesovist did not affect the proliferation and viability of rat bone marrow mesenchymal stem cells, while 50-100g/m L Roesovist inhibited the proliferation of R BMMSCs, slowed cell cycle and promoted cell apoptosis, indicating that 25g/m L Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/m L Roesovist did not affect the osteogenesis of R BMMSCs. The third part of the study shows that the magnetic field can simulate the target migration of SPIO-labeled stem cells under the magnetic field in vitro. In vivo, a magnetic targeted induction cell homing experiment of alveolar bone defect was constructed, and the results showed that the long-distance migration of the cells labeled with SPIO could be simulated perfectly. Magnetic field can promote the implantation of SPIO-labeled stem cells in alveolar bone defects. Whether the implanted stem cells can continue to proliferate, differentiate into new bone cells and then participate in the weight of alveolar bone Jian Shang needs further in-depth study to prove it.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781
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本文編號(hào)：2234554